Boehringer Mannheim Gmbh

Hans-Otto Beutler

Stephan Fischer

Ulfert Deneke

Herwig Brunner

Gunter Lang

Klaus Kaluza

Georg-Burkhard Kresse

Walter Schneider

Barbel Wurst

Ingrid Schutte

Gerhard Micharl

The invention concerns a new NAD.sup.+ -dependent D-malate dehydrogenase (decarboxylating, E.C. 1.1.1.83) from E. coli which consists of two subunits which each have a molecular weight of about 42 kD (SDS gel) and processes for the isolation of this enzyme from E. coli and for its stabilisation. The invention concerns in addition a method for the determination of D-malate with the D-malate dehydrogenase according to the present invention as well as a reagent for determining D-malate.

Enzymatic reagent for D-malate determination

The present invention provides a stabilized, NAD(P)H-dependent, soluble nitrate reductase of the assimilatory type, characterized by a molecular weight of about 90,000 D in the case of electrophoresis in the presence of sodium dodecyl sulphate and a residual activity after 3 weeks at 35.degree. C. of more than 60%, obtainable by preparing a suspension of the comminuted starting material in tris/tartaric acid buffer (pH 7-8.5), adding soluble polyethyleneimine thereto in such an amount that 10 to 20% of the nitrate reductase activity passes into the precipitation obtained, separating off the precipitate and working up the supernatant according to conventional biochemical methods of fractionation in the above-mentioned digestion buffer, dialyzing the enzyme solution obtained and lyophilizing in a zwitterionic buffer.

Stabilized, NAD(P)H-dependent, soluble nitrate reductase, a process for

In a method for the quantative enzymatic determination of adenosine diphosphate (ADP) in aqueous solution by phosphorylation of the ADP with a kinase in the presence of a phosphorylated substrate therefor, with the formation of ATP and a dephosphorylated substrate, such as galactose, formate, pyruvate or nucleoside diphosphate, the dephosphorylated substrate is reacted, optionally with the insertion of a further adjuvant reaction, such as a decarboxylation, transamination or hexosidiation, with NAD(P) in the presence of a dehydrogenase and the NAD(P)H formed is either measured directly or reacted with a tetrazolium salt in the presence of an electron carrier, with the formation of a formazane, which latter is then measured. A reagent suitable for carrying out this method contains a kinase, a phosphorylated substrate therefor different from ADP, a specific dehydrogenase for the dephosphorylated form of the substrate or for a transaminated, decarboxylated or hexosidized secondary product thereof, the co-enzyme of the dehydrogenase and a buffer, as well as optionally a tetrazolium salt, an electron carrier suitable therefor and optionally a surface-active agent.

Method for the quantitative enzymatic determination of ADP

The present invention provides a process for the enzymatic determination of sulphite, wherein the sulphite is oxidized in a buffered solution with sulphite oxidase to give sulphate, the hydrogen peroxide thereby formed being reduced in the presence of NAD and NADH with the enzyme NADH peroxidase and the consumption of NADH being determined optically. The present invention also provides a reagent for the enzymatic determination of sulphite, wherein it contains sulphite oxidase, NADH, NAD, NADH peroxidase and buffer.

Process and reagent for the enzymatic determination of sulphite

Growing community of inventors

Tutzing, Germany

Hans-Otto Beutler